FIGURE 3

FIGURE 3

Figure 3. Zebrafish U-box^{− /−} expressing truncated Chip with impaired ubiquitin ligase activity were generated by CRISPR/Cas9. Zebrafish stub1 gene was targeted for mutagenesis at the last exon (A), leading to an introduction of 7 bp-deletion mutation in this exon (c.761-769del) which was detected by MluI restriction digestion (B) and DNA sequencing (C). The 761-769del mutation is predicted to result in a frameshift and early stop codon and truncation of C-terminal U-box domain (D). Recombinant Chip proteins were produced from the cDNA of U-box^{− /−} and wild-type zebrafish, and further examined for in vitro ubiquitin ligase activity on Chip (E, lane 1–3) and Hsc70 substrates (E, lane 4–6). Compared to the wild-type human CHIP (E, lane 1 and 4) and wild-type zebrafish Chip (E, lane 2 and 5) controls, U-box truncated zebrafish Chips showed impaired self-ubiquitination (E, lane 3) and Hsc70 ubiquitination activities (E, lane 6).